Báo cáo hóa học: "Generalized cerebral atrophy seen on MRI in a naturally exposed animal model for creutzfeldtjakob disease"

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McKnight et al. Journal of Translational Medicine 2010, 8:125 http://www.translational-medicine.com/content/8/1/125 RESEARCH Open Access Generalized cerebral atrophy seen on MRI in a naturally exposed animal model for creutzfeldt- jakob disease Alexia L McKnight1*†, Lawrence A Minkoff2†, Diane L Sutton3, Bruce V Thomsen4, Perry L Habecker5, Raymond W Sweeney6, Gary Smith7, Constantin A Dasanu8, Thomas E Ichim9, Doru T Alexandrescu10, Joel M Stutman11† Abstract Background: Magnetic resonance imaging has been used in the diagnosis of human prion diseases such as sCJD and vCJD, but patients are scanned only when clinical signs appear, often at the late stage of disease. This study attempts to answer the questions “Could MRI detect prion diseases before clinical symptoms appear?, and if so, with what confidence?” Methods: Scrapie, the prion disease of sheep, was chosen for the study because sheep can fit into a human sized MRI scanner (and there were no large animal MRI scanners at the time of this study), and because the USDA had, at the time of the study, a sizeable sample of scrapie exposed sheep, which we were able to use for this purpose. 111 genetically susceptible sheep that were naturally exposed to scrapie were used in this study. Results: Our MRI findings revealed no clear, consistent hyperintense or hypointense signal changes in the brain on either clinically affected or asymptomatic positive animals on any sequence. However, in all 37 PrPSc positive sheep (28 asymptomatic and 9 symptomatic), there was a greater ventricle to cerebrum area ratio on MRI compared to 74 PrPSc negative sheep from the scrapie exposed flock and 6 control sheep from certified scrapie free flocks as defined by immunohistochemistry (IHC). Conclusions: Our findings indicate that MRI imaging can detect diffuse cerebral atrophy in asymptomatic and symptomatic sheep infected with scrapie. Nine of these 37 positive sheep, including 2 one-year old animals, were PrPSc positive only in lymph tissues but PrPSc negative in the brain. This suggests either 1) that the cerebral atrophy/neuronal loss is not directly related to the accumulation of PrPSc within the brain or 2) that the amount of PrPSc in the brain is below the detectable limits of the utilized immunohistochemistry assay. The significance of these findings remains to be confirmed in human subjects with CJD. Background fatal neurodegenerative diseases that have very long Scrapie was first reported in 1730 in sheep and goats incubation periods and unique neuropathological and is the longest known transmissible spongiform changes. The most widely accepted cause of the TSE encephalopathy (TSE) [1]. In the past two decades, TSEs diseases is an abnormal prion protein, identified as PrPSc in the case of scrapie, which is a stereoisomer of have received much attention since ingestion of bovine the normal prion protein (PrPC). spongiform encephalopathy (BSE) infected beef was cau- sally linked to the variant form of CJD (vCJD) [2]. These Ante-mortem diagnosis of the TSE diseases, in gen- TSE diseases are progressively debilitating and invariably eral, has proven to be quite challenging. MRI has been useful in CJD patients – with both the sporadic and var- * Correspondence: alexia@mcknightinsight.com iant forms. It is helpful in the exclusion of other neuro- † Contributed equally degenerative diseases as well as, in some cases, the 1 Assistant Professor of Radiology, University of Pennsylvania School of positive diagnosis of sCJD or vCJD [3-6]. For example, Veterinary Medicine, New Bolton Center, Kennett Square, PA 19348, USA Full list of author information is available at the end of the article © 2010 McKnight et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
McKnight et al. Journal of Translational Medicine 2010, 8:125 Page 2 of 8 http://www.translational-medicine.com/content/8/1/125 i n a study of 162 sCJD cases, bilateral basal ganglia scrapie flock in the Midwest United States that was to hyperintensity was found to be 67% sensitive and 94% be depopulated for regulatory reasons. The flock was specific. MRI findings included hyperintense alterations comprised of 62 black faced breeds (24 Hampshire, 39 and/or brain atrophy, alone or in combination with each Suffolk, 1 unknown black faced), 37 Western white other. A normal MRI without any hyperintense changes faced sheep which entered the flock as adults prior to or atrophy was seen in 27.2% (44/162) patients [3]. A this study, and 12 brockel faced sheep born of the white bilaterally symmetric hyperintense pulvinar, or the faced ewes and black faced rams. The sheep ranged in “hockey stick sign”, is reported to be present in 80% of age from 1-9 years old with the oldest sheep primarily vCJD patients in some studies [4,5,7]. CJD patients, the Western white faced breed. however, present with clinical symptoms at a relatively Six additional sheep were purchased from two sepa- late stage of the disease. rate certified scrapie free flocks; five were black faced MRI abnormalities are reported in pre-symptomatic and one was white faced. They ranged in age from 2-8 mice experimentally infected (intraperitoneally) with years and served as known negative controls. scrapie. The study was performed at 9.4T, and a hyper- intense septum and hippocampus were seen at 120 days MRI Examinations - Part I post infection, approximately 60 days prior to the onset Brain MR examinations were initially done on the 6 of clinical signs. Additional cortical and thalamic negative control animals and the 24 scrapie positive abnormalities were seen at 180 days post infection, sheep which were identified by an immunohistochemis- try test of surgically collected 3 rd eyelid lymph tissue when clinical signs became apparent [8]. Other MRI studies by Chung et al. in rodent scrapie from the 76 black and brockel faced sheep [12,13]. Each models correlate MRI signal changes to neuoropathol- animal was scanned live under general anaesthesia and ogy [9,10]. One hamster model, performed at 4.7T, with then recovered with the exception of the three most scrapie induced intra-cerebral injections revealed a cor- clinically affected animals, which were euthanized after relation with increased T2 signal and gliosis, and the MRI exam, and one sheep that died during induc- decreased T2 signal with vacuolization. In some areas tion. We used a mobile 1T GE Signa LX MRI system with marked gliosis and vacuolization, no MRI signal (General Electric, Milwaukee, WI) with the general pur- changes were seen suggesting a T2 cancelling effect [10]. pose flex coil wrapped around the dorsal and lateral In contrast to Chung ’ s findings in hamsters, Haik aspects of the head. The following pulse sequences were et al. found no association of MRI signal change in two obtained: T1- and T2-weighted fast spin echo, proton CJD patients with gliosis and no clear association with density (PD), inversion recovery (IR), fluid attenuated spongiform change. There was, however, strong correla- inversion recovery (FLAIR), and diffusion weighted ima- tion of MRI signal change with accumulation of PrPSc in ging (DWI). The slice parameters were 3 mm thickness, the both the sCJD and the vCJD patient [11]. 0 gap, 14 cm FOV for PD, T1, T2, IR, and FLAIR; for Unlike experimentally induced scrapie rodent models DWI 4 mm thickness, 0 gap, 22 cm FOV. Where possi- that have a different course of disease than natural ble 23 slices in the each of the axial, sagittal, and coro- infection and CJD patients that present with an nal planes were obtained. advanced stage of disease, a naturally exposed scrapie flock is typically composed of sheep in various stages of MRI Examinations - Part II disease. For this reason, these animals are considered a Based on the findings from Part I, all 113 remaining good model to study MRI findings in scrapie as a model sheep were examined or re-examined by the same MRI for the TSE diseases. Our objective was to study the protocol (with the exception of the T1 and FLAIR consistent MRI findings in a large flock of scrapie posi- sequences) immediately following euthanasia. tive animals as confirmed by immunohistochemistry. The purpose was twofold: 1) to better understand TSE Quantitative Analysis diseases by evaluating the MRI finding in naturally Lateral ventricle to cerebrum area ratios (V/C ratio) infected sheep, and 2) to assess the accuracy of MRI in were calculated in all sheep. The V/C ratio is calculated the detection of TSE in both symptomatic and asympto- by the following formula: matic sheep. Lateral ventricle to cerebrum Ratio = Lateral ventricle area / (cerebrum area − lateral ventricle area) * 100 or, Methods more concisely: V / C = A V /(A C -A V ) * 100 and is reported as a percent. Flock information It is the area of the lateral ventricle normalized to the One hundred eleven scrapie-exposed sheep with the area of the sheep ’ s cerebrum area as imaged in that scrapie susceptible QQ 171 genotype were used in this sagittal slice, and the value is reported as a per cent. It study. The sheep originated from a single commercial
McKnight et al. Journal of Translational Medicine 2010, 8:125 Page 3 of 8 http://www.translational-medicine.com/content/8/1/125 i s an effort to measure the size of this sheep ’ s lateral this required a line drawing rule over that narrow ventricle area as related to its own cerebrum area (with- region, which rule was used by both. Resulting percen- out the ventricle area included in the cerebrum area). tages were similar, only varying by a small multiplicative On the FSE T2 weighted sequences, a sagittal slice 3-5 constant; and, finally, both obtained similar graphs. mm off midline that had the largest lateral ventricular For the total data set of 117 sheep, the inter-observer area was used for the measurements. As shown in Fig- reliability (correlation coefficient) between the scientist ure 1, regions of interest were drawn around the border A and scientist B was 0.85 by the Pearson Product of the lateral ventricle and, in the same slice, around the Moment Method and 0.87 by the Spearman Rank Order cerebrum. Method. Two scientists measured the areas using two different methods on different computers with no communica- Laboratory analysis tion between them regarding their results. The scientists Scrapie testing by immunohistochemistry procedures did their work in separate locations at different times followed the standard protocols used in the United with no communication regarding the V/C results. Both States Department of Agriculture (USDA) scrapie eradi- used mouse pointers to trace the outlines of the lateral cation program and are similar to those described pre- ventricle and the cerebrum in the same slice as defined viously [13]. The pre-mortem third eyelid tissues were above. evaluated at the University of Wyoming (EW) and post- Scientist A used Adobe’s Photoshop software and their mortem sections of medulla at the obex, medial retro- “Magnetic Lasso” technology with the following para- pharyngeal lymph node and tonsil were examined at the meters: feather = zero pixels; anti-aliased = on; width = National Veterinary Services Laboratory in Ames, IA 3 pixels; edge contrast = 100%, and frequency = 100. (BT). Briefly, tissue sections were deparaffinized, rehy- Scientist B wrote his own software code in Microsoft drated, treated with 95% formic acid (lymph tissue only) Visual Basic 6.0 with DicomObjects.ocx as the DICOM and then autoclaved in an antigen retrieval solution interface and created a routine which counts pixels obtained from DakoCytomation, Carpinteria, CA, USA inside bounded planar regions. Dicomobjects.ocx is a [14]. The sections were stained with an automated library of compiled software enabling DICOM files to be immunohistochemistry system (by Ventana Medical Sys- studied using many different higher level programming tems, Tucson, AZ USA) which used a mixture of two languages for control. http://www.medicalconnections. monoclonal antibodies, F89/160.1.5 and F99/97.6.1, to co.uk. detect prion protein [13]. Known positive and negative Both techniques used exactly the same definition of tissue samples were run as controls for each group of the boundary between the cerebrum from the cerebel- slides. The slides were interpreted independently of the lum, the only place in the slices of interest in which the MRI results. Later, additional areas of the brain (4 areas boundary was less clear than all other tissue boundaries: of the cerebrum and l section each of the thalamus, ros- tral colliculus, pons and cerebellum) were examined by IHC on eight of the nine animals which were found to be positive only on lymph tissue and negative on brain samples at the level of the obex. All sheep were genotyped at codons 171, 136, and 154. There were only 4 genotypes present in the 117 sheep: 97 AARRQQ; 4 AARHQQ; 4 AARRQR; and 12 AVRRQQ. Clinical examinations All 111 scrapie exposed sheep were evaluated clinically for neurological signs consistent with scrapie. The 24 eyelid positive animals were more thoroughly examined by recording the following parameters: body condition score, percent wool loss, presence of ataxia, and trembling. Figure 1 The quantitative analysis in this study was performed by outlining the perimeter of the lateral ventricle and the Statistical analysis cerebrum on a sagittal slice 3-5 mm from midline where the largest area of lateral ventricle was present. The areas were The extent to which the MRI results discriminate determined and the ventricle to cerebrum ratio (V/C ratio) was then between “scrapie” and “not scrapie” was evaluated using calculated. a receiver operating characteristic (ROC) curve
McKnight et al. Journal of Translational Medicine 2010, 8:125 Page 4 of 8 http://www.translational-medicine.com/content/8/1/125 c onsisting of a graph of sensitivity versus one minus AARRQR; and 1 of 12 AVRRQQ. Every score above 10.4% corresponded to a PrPSc positive and every score specificity as the cutoff is varied. The parameters and characteristics of the ROC curve was estimated from the below 9.5% corresponded to a negative: there was only a data using STATA (Statacorp, 2001). The area under 10.25% overlap in scores, and most importantly only the ROC curve is used as a summary measure of the 8.75% false negatives when all scores are considered and extent of the discrimination [15]. the above cutoffs are not used. In addition, it is noteworthy that the AVRRQQ sub- jects are, despite their relatively small N, the most Approvals All aspects of this study were approved by the Univer- ambiguous, in that 5 of the 11 negatives with this geno- sity of Pennsylvania’s Institutional Animal Care and Use type fall into the upper quartile of all negative scores; this ‘leaning’ towards the high end of the negative distri- Committee, Environmental Health and Radiation Safety, and the Pennsylvania Department of Agriculture. bution, might serve to suggest that over time this geno- type might turn out to be the most likely to shift from Results negative to positive, and in future work should receive The clinical signs of scrapie, trembling and ataxia with special attention regarding possible false negatives. various combinations of wool loss and/or thin body con- The ROC curve is shown in Figure 5. The area under dition score, were only identified in 9 sheep. The other the curve was 0.99 (95% confidence interval, 0.98-1.00). 102 sheep in the infected flock showed no detectable As described in Hosmer and Lemeshow (page 162), this is in the “outstanding discrimination” range [15]. signs consistent with scrapie. IHC testing found 37 out of 111 sheep positive for scrapie with 9 of 37 sheep Discussion positive only on lymph tissues. Eight of these 9 lymph- only positive sheep remained PrPSc negative following The 111 scrapie exposed QQ sheep used in this study additional IHC testing on multiple areas of the brain. are from a single commercial flock in the Midwest Uni- Additional brain samples were unavailable for testing on ted States that had a high prevalence of infection (33% of the QQ animals were PrPSc positive on post-mortem the single remaining animal. The third eyelid test identified 24/76 (31%) sheep as IHC). Because there was a wide range in ages, multiple PrP Sc positive. Performing the eyelid test allowed an breeds, and clinical stages of the disease progression, antemortem diagnosis to identify several scrapie infected this flock was considered a good model for evaluating sheep. In Part I of the study when brain MRI exams of MRI findings in scrapie positive sheep. The MRI find- these eyelid positive animals were compared to the 6 ings correlated with IHC results in each of the breeds control animals, no clear, consistent MRI signal changes examined. The scrapie associated MRI changes detected were noted in the brain of either the 9 clinically affected subclinically infected animals and also detected 10 ani- or the 15 asymptomatic sheep on any pulse sequence. mals which were not identified by the current antemor- As seen in Figure 2, the most severely affected clinical tem third eyelid test. animals had hyperintense adipose tissue, predominantly MRI signal abnormalities were not seen consistently within the medullary cavity of the skull and around the on T2, FLAIR, or PD weighted images in this flock as retropharyngeal lymph nodes, corresponding with serous reported in CJD patients and rodent scrapie models. atrophy secondary to emaciation. There was also mild Although similar inconsistencies are also seen in people, subjective enlargement of the lateral ventricles with sul- hyperintense changes in this study were a rare finding. cal prominence in the most clinically affected sheep Reasons for the hyperintense change in some animals indicative of diffuse cerebral atrophy (Figure 2). This and not others remain unclear. Meissner et al. found a finding prompted quantitative evaluation in all sheep. correlation between the presence and absence of MRI The results of the quantitative analysis following Part findings and the CJD genotype in human patients [6]. II of the study are shown in Figures 3 and 4. The 37 The genotypes of this flock are very homogeneous and PrPSc positive sheep had larger V/C ratios compared to could explain the relative uniform lack of MRI signal the PrPSc negative sheep (Figure 3). Interestingly, 9 of abnormality. these 37 sheep, including 2 one-year olds, were PrPSc The animals with the most severe clinical signs had positive in the retropharyngeal lymph nodes and/or ton- the highest ventricular to cerebrum ratios; almost all sils but negative in the brain (Figure 4). As seen in Fig- animals with a V/C ratio over 15% showed clinical ure 4, no correlation with the V/C ratios with age was symptoms of scrapie. For this reason, we believe that seen. Almost all animals with a V/C ratio over 15% enlarged ventricular to cerebral ratios may be positively showed clinical symptoms of scrapie. correlated with disease progression. Similar correlations The 37 PrP Sc positive sheep fall into the following between brain atrophy as seen on MRI and progression genotypes: 36 of 97 AARRQQ; 0 of 4 AARHQQ; 0 of 4 of clinical disease have also been reported in other
McKnight et al. Journal of Translational Medicine 2010, 8:125 Page 5 of 8 http://www.translational-medicine.com/content/8/1/125 Figure 2 Axial and sagittal MR images of a normal control sheep (A and B) compared to the most clinically affected animal in the study (C and D). The sulcal prominence and enlarged lateral ventricles indicative of diffuse cerebral atrophy are seen in the scrapie affected sheep. neurodegenerative diseases such as Alzheimer’s disease such as the V/C ratio as used in this study, may be con- [16] and multiple sclerosis [17,18]. sidered as an ante-mortem tool for live animals at risk Ventricular enlargement with sulcal prominence is for scrapie, including young animals. typical of brain atrophy on MRI examinations The pathophysiologic process that would explain dif- [16,19,20]. This observation in the most clinically fuse cerebral atrophy in young asymptomatic sheep is affected animals suggested similar evidence of cerebral unclear. The progression of scrapie in the naturally atrophy/neuronal loss that has been reported in CJD infected animal begins with an oral infection. Particu- patients[3] and a rodent scrapie model[10] with larly susceptible in the perinatal period, lambs first show evidence of PrPSc in the Peyer’s patches, medial retro- advanced disease. Particularly noteworthy was the find- ing, following quantitative analysis of all 117 sheep (111 pharyngeal lymph nodes, mesenteric lymph nodes, and scrapie exposed and 6 normal controls) that cerebral tonsils about 2-5 months after birth [21-23] In approxi- atrophy was a consistent finding in the 37 PrPSc animals, mately 12-18 months, but as early as 9-10 months, PrPSc enters the central nervous system and can be first even among the asymptomatic sheep and, of particular interest, in the 2 positive one-year old sheep. The quan- found in the obex of the medulla and the T8-T10 thor- tification of certain brain parameters on MR images, acic spinal cord segments [22,24] At the terminal stage
McKnight et al. Journal of Translational Medicine 2010, 8:125 Page 6 of 8 http://www.translational-medicine.com/content/8/1/125 Histogram of Per Cent Ventricle Area to Cerebrum Area 10 9 8 7 Number of Sheep 6 5 * 4 * 3 * 2 ** 1 ** ** 0 0% 5% 0% 5% 0% 5% 0% 5% 0% 5% 0% 5% % % % % % % % % % % % % % % % .0 .5 .0 .5 .0 .5 .0 .5 .0 .5 .0 .5 .0 .5 .0 4. 4. 5. 5. 6. 6. 7. 7. 8. 8. 9. 9. 10 10 11 11 12 12 13 13 14 14 15 15 16 16 17 Ratio Ventricle to Cerebrum (%) Control Negative SCRAPIE Figure 3 The quantitative results of the study are displayed in this histogram. The 37 PrPSc positive sheep (red) have larger V/C ratios relative to 74 PrPSc negative sheep (yellow) and 6 normal controls (blue) as defined by immunohistochemistry. Nine of the 37 positive sheep showed clinical signs of scrapie (asterisks). Age of Sheep vs Per Cent Ventricles 18.00% --PrPSc negative in the brain 16.00% 14.00% % of Ventricle to Cerebrum 12.00% 10.00% 8.00% 6.00% 4.00% 0 1 2 3 4 5 6 7 8 9 10 Age (years) SCRAPIE NEGATIVE Controls Figure 4 No correlation of the lateral ventricle to cerebrum ratio with age was seen, only larger ratios in PrPSc positive sheep as displayed in red. Nine of the 37 positive sheep, depicted by arrows, were PrPSc negative in the brain, but positive in the lymph tissue, including 2 one-year old animals.
McKnight et al. Journal of Translational Medicine 2010, 8:125 Page 7 of 8 http://www.translational-medicine.com/content/8/1/125 that the diffuse cerebral atrophy in this scrapie flock is ROC Curve (Receiver Operating Characteristics) either not directly related to the accumulation of PrPSc 1.20 in the brain or that low levels of PrPSc in the obex are present but simply undetected by the IHC testing. 1.00 Conclusions We found a greater lateral ventricle to cerebrum area Area under the ROC curve = 0.99 True Positive Rate (Sensitivity) 0.80 ratio in PrPSc positive sheep compared to PrPSc negative sheep in this large, naturally scrapie infected flock. There was no age correlation with the V/C ratios, only 0.60 higher ratios in more clinically affected sheep in advanced disease. The results of this study indicate that 0.40 there is diffuse cerebral atrophy/neuronal loss seen with MRI in naturally infected scrapie sheep in young ani- mals and prior to the onset of clinical signs that appears 0.20 related to the progression of the disease. These results also suggest that the cerebral atrophy/neuronal loss is not directly related to the accumulation of PrPSc within 0.00 0.00 0.20 0.40 0.60 0.80 1.00 the brain, or that the amount of PrPSc in the brain is False Positive Rate (1-Specificity) below the detectable limits of the immunohistochemis- Figure 5 ROC curve shows the V/C ratio is in the ‘outstanding try assay. The significance of these findings remains to discrimination’ range for correctly characterizing scrapie be confirmed in human subjects with CJD. positive animals in this highly infected scrapie flock. of disease in clinically affected animals, usually between Acknowledgements The authors would like to acknowledge Dr. Elizabeth Williams at the 2-5 years of age, neuropil vacuolation, astrocytosis, neu- Wyoming State Veterinary Laboratory for evaluating the third eyelid tissues, ronal loss, and shrunken ‘ dark neurons ’ are seen in and Patricia Meinhardt and Norma Newton from National Veterinary Services areas rostral to the medulla [25]. These histopathologi- Laboratories in Ames, Iowa for the genotypic evaluations. The authors heartily thank the faculty and staff of New Bolton Center’s cal findings are reported to be within the neocortex of Anesthesia and Pathology, as well as the MRI technologists and farm the cerebrum (particularly centered around the superior assistants for all their help with this study. Nigel Watson of the New Bolton frontal lobe gyrus) as well as the diencephalon and Pathology Department who did consistently precise dissection work on the obex and nodes of all 117 sheep deserves special recognition. mesencephalon, cerebellum, and brain stem [25]. Acknowledgement is gratefully tendered to Dr. David Harvey, General However, as shown in the pathogenesis studies, PrPSc Manager of Medical Connections, UK for very intelligent software assists with within the cerebrum in asymptomatic sheep is not an Dicomobjects.ocx. The authors are very grateful for the insightful statistical assistance, relative to the genotype analysis as well as the inter-observer expected finding [21,22,24,25]. Routine screening for scrapie reliability, provided by Dr. Leonard A. Rosenblum (Professor of Experimental on IHC is performed at the level of the obex of the medulla, Psychology (Retired), Downstate Medical Center, Brooklyn, NY). because identification of PrPSc at this site has shown to be Thank you also to the Louis and Lena Minkoff Foundation for the generous gift that funded this project. Dr. Lawrence Minkoff is the president of the most sensitive for detection of scrapie in the earliest stages Foundation and an author of this paper. of CNS involvement [26,27]. Ersdal et al. found evidence of PrPSc in the cerebellum of 1/17 sheep and not in the obex Author details 1 [24]. Accumulation of PrPSc, spongiform change and/or Assistant Professor of Radiology, University of Pennsylvania School of Veterinary Medicine, New Bolton Center, Kennett Square, PA 19348, USA. astrogliosis rostral to the obex in asymptomatic sheep was 2 Executive Vice President, Fonar Corporation, Marcus Drive, Melville, NY, USA. not reported and considered to be unusual [24,26-30]. 3 National Scrapie Program Coordinator, United States Department of Agriculture, Animal and Plant Health Inspection Service, Veterinary Services, Therefore, diffuse cerebral atrophy in the 28 asymptomatic Riverdale, MD 20737, USA. 4United States Department of Agriculture, Animal sheep in this study is difficult to explain. and Plant Health Inspection Service, Veterinary Services Laboratories, Ames, Furthermore, 9 of the 37 PrPSc positive sheep in this IA 50010, USA. 5Professor, The Pennsylvania Animal Diagnostic Laboratory study that had higher V/C ratios compared to PrP Sc System at New Bolton Center and the Laboratory of Pathology and Toxicology, School of Veterinary Medicine, University of Pennsylvania, 382 W. negative sheep were only PrPSc positive in lymphoid tis- Street Road, Kennett Square, Pennsylvania 19348, USA. 6Professor of sue, not in the obex. Additional immunohistochemistry Medicine & Chief, Section of Medicine, Department of Clinical Studies, New Bolton Center, 382 West Street Road, Kennett Square, PA 19348, USA. was negative on areas of the brain rostral to the obex in 7 Professor of Population Biology and Epidemiology & Chief, Section of the 8 re-tested animals, which is consistent with the lit- Epidemiology and Public Health, Department of Clinical Studies, New Bolton erature that the obex negative PrP Sc sheep are in an Center, 382 West Street Road, Kennett Square, PA 19348, USA. 8Saint Francis Hospital and Medical Center, Hartford, CT 06105, USA. 9Medistem Inc., San early stage of disease that has not yet reached the cen- Diego, CA 92101, USA. 10Georgetown Dermatology, Washington, DC 20010, tral nervous system [22]. Two possible explanations are USA. 11Professor and Chairman (Retired), Medical Computer Science
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